The main objective of this proposal is to study the kinetics of uptake and intracelluar accumulation of labelled sphingolipids fed to human fibroblasts in culture derived from GM2-gangliosidosis, GM1-gangliosidosis, Krabbe's, Gaucher's and Niemann-Pick diseases. In separate experiments, the endogenous sphingolipids of these cells will be labelled with radioactive precursors in the medium to understand better the dynamic aspects of metabolic disturbances occurring in these disorders. The gangliosides and neutral glycosphingolipids will be analyzed by very sensitive high performance liquid chromatographic methods developed in this center. Experiments are designed to permit direct insight into what does happen in the cell by looking for metabolic defects in situ as well as correction in situ by providing the appropriate missing enzyme to the culture medium of these fibroblasts. This study can help to diagnose the variant forms of these sphingolipidoses arising from different degrees of metabolic derrangement caused by heterogeneity in the genetic mutation of the deficient enzyme. The AB variant form of GM2-gangliosidosis can be clearly diagnosed by following the accumulation and metabolism of GM2 in the fibroblasts from this disorder. The role of a specific nonenzymic factor in correcting GM2 accumulation in the AB variant fibroblasts will be evaluated. Lactosyl ceramide metabolism will be studied in fibroblasts from Krabbe's disease and GM1-gangliosidosis to determine whether a true metabolic defect for lactosyl ceramide hydrolysis exists in situ in these disorders or whether the enzymic deficiency observed in vitro is an artifact of in vitro assay technique.